Abstract Cytotoxic T cells play a central role in tumor immunity based on their ability to target tumor cells with a high degree of specificity. Checkpoint blockers that target the inhibitory CTLA-4 and PD-1 receptors on T cells have made a major impact on the treatment of melanoma and other human cancers. However, primary and secondary resistance to checkpoint blockade remain major clinical problems, and most melanoma patients still fail to benefit from these therapeutic agents. The goal of this project is to define the transcriptional and epigenetic mechanisms that render melanomas resistant to T cell-mediated immunity. We recently performed a genome-scale screen and discovered a large number of genes (n=128) that mediate resistance by melanoma cells to cytotoxic T cells. Of particular interest are three major transcriptional and epigenetic regulators, the PBAF form of the SWI/SNF complex as well as the SOX4 and NF-kB p65 transcription factors. Aim 1 will focus on the role of PBAF complex in resistance to immunotherapy. The PBAF complex is relevant in human melanoma because the ARID2 gene is frequently mutated. Preliminary data demonstrate that inactivation of the genes encoding the three unique subunits of the PBAF complex (Pbrm1, Arid2 and Brd7) sensitizes murine melanoma cells to cytotoxic T cells. We will investigate the impact of the PBAF complex on the tumor microenvironment in human melanoma as well as murine and zebrafish models of melanoma. Mechanistic studies will focus on the epigenetic enzymes and transcription factors that cooperate with the PBAF complex to inhibit T cell-mediated tumor immunity. Aim 2 will focus on the two major transcription factors ? NF-kB p65 and SOX4 ? that were discovered in the genetic screen. We will investigate whether the NF-kB and SOX4 transcription factors cooperate with the PBAF complex to confer resistance to cytotoxic T cells. The TNF??? NF-kB pathway was identified as the top resistance pathway in the screen, and we will investigate whether TNF? secreted by activated T cells renders neighboring tumor cells more resistant to T cell-mediated killing. The TNF? ? NF-kB pathway can induce either cell survival or cell death, and a number of gene products identified in the screen inhibit TNF? induced cell death, including the kinase TBK1. We will therefore investigate whether the outcome of TNF?-mediated signaling can be switched from tumor cell resistance to cell death using available TBK1 inhibitors. Expression of the transcription factor SOX4 is induced by TGF? following activation of latent TGF? by integrin ?V. Both genes encoding SOX4 (Sox4) and integrin ?V (Itgav) were identified as major resistance mechanisms in our genetic screen, and we will investigate whether this resistance pathway can be targeted with a mAb that inhibits integrin ?V mediated TGF? activation. This project will make a substantial contribution to this P01 through major collaborations with Drs. Fisher and Zon (melanoma models), Drs. Rodig, Hodi and Fisher (human melanoma) and Drs. Liu, Zon and Fisher (epigenetic mechanisms).